Gene transfer to airway epithelia has the potential to become an important new treatment for cystic fibrosis (CF) lung disease. However, the poor efficiency of binding and infecting differentiated airway epithelia is a major barrier for adenovirus, as well as for most other vectors. This proposal builds on our earlier work to answer the specific aims. 1) What is the role of the serotype 2 adenovirus (Ad2) receptors in adenovirus infection of human airway epithelia? Our earlier work indicated that the apical membrane of differentiated human airway epithelia lacks receptor activity for adenovirus fiber. With the recent identification of type 2/5 adenovirus receptors, CAR and MHC class Ia2, it is now possible to investigate the cellular and molecular bases for limited adenovirus type 2/5 binding. The results of these studies will tell us why adenovirus infection is relatively inefficient and will help us enhance gene transfer with adenovirus and other vectors. 2) Will genetic modifications of the adenovirus fiber increase vector binding and infection of airway epithelia? In preliminary studies we discovered that serotype 17 adenovirus (Ad17) bound to and infected differentiated airway epithelia more efficiently than Ad2 and Ad5 serotypes, which are currently used as vectors. We will investigate the mechanisms responsible for increased binding and infection. Our preliminary data with a chimeric type 2 adenovirus expressing Ad17 fiber are very encouraging; we will test the hypothesis that this novel chimeric vector can enhance gene transfer to human airway epithelia and correct the CF defect. 3) Will provide modifications of the adenovirus fiber protein improve binding and infection? Our preliminary data and earlier work suggest that if adenovirus binding to the apical surface of airway epithelia can be improved, then gene transfer will be increased. To find new ligands for the unknown apical receptors, we will build on our preliminary work using phage display libraries. This combinatorial approach will identify novel peptide ligands which bind to and are internalized by the apical membrane. We will insert the peptides into the adenovirus fiber protein of recombinant adenovirus fiber protein of recombinant adenovirus vectors and investigate their interaction with the epithelium and ability to enhance gene transfer. These studies using adenovirus vectors will increase our understanding of the mechanisms of gene transfer, improve knowledge of interactions between vectors and the airways, and will suggest new approach to enhance efficiency. Importantly, the results will have immediate application to new generations of adenovirus vectors, as well as other vectors.